The heat shock proteins (hsps) are synthesized by a host cell in response to physical, chemical or biological stresses. A typical example of a biological stress includes infection of a host cell with a virus.
One member of hsps is the family of proteins known as hsp70. The function of hsp70 is to assist in thermotolerance, prevention of misfolding of nascent polypeptides, transmembrane protein transport and nuclear protein transport (Welch, 1993).
A virus that induces the expression of hsp70 in humans is the human cytomegalovirus (CMV). CMV is a member of the herpesvirus family.
Expression of hsp70 may be induced by several CMV proteins that activate the hsp70 promoter. These CMV proteins include the CMV immediate-early proteins 1 (IE1) and 2 (IE2), and the US3 and UL37 gene products (Hagemeir et al., 1992). The hsp70 protein has no known function in the virus replication cycle, but theoretically may assist in assembly of the CMV virion.
CMV is the most common viral intrauterine infectious agent, affecting 0.5-2.5% of all live births (Hagay et al., 1996). The infectious virus reaches the uterus via the bloodstream. The virus then traverses the placenta and infects the developing fetus. Such fetal CMV infections are called congenital infections.
Transmission of the virus to the fetus may occur as a result of either a primary maternal CMV infection or a recurrent maternal CMV infection. A primary maternal CMV infection is the initial infection of the mother with CMV. In contrast, a recurrent infection occurs as a result of reactivation of endogenous latent CMV or a reinfection with a new strain of CMV.
The transmission of CMV to a fetus may occur any time throughout a pregnancy. Primary maternal infection poses the major risk for congenital infection with a transmission rate of approximately 30-40%. The rate of transmission for a recurrent infection is about 0.15-1%.
Ten percent of congenitally infected infants develop congenital CMV syndrome (eg., death, encephalitis, neurological problems, etc.) while 90% are asymptomatic at birth (Hagay et al., 1996). Of the 10% of congenitally CMV infected infants, approximately 20% die and 72% develop major neurological problems.
Of the 90% of congenitally infected infants that are asymptomatic at birth, 5-7% will be afflicted by late sequelae. The late sequelae include mental retardation, deafness, and hearing defects. These symptoms usually appear during the first two years of life (Demmler 1994; Stagno et al., 1986).
Diagnosis of congenital CMV infection is typically made by detecting CMV in the amniotic fluid by culture or polymerase chain reaction (PCR). This method yields a sensitivity of about 70-100% (Hagay et al., 1996; Levy et al., 1996; Bodeus et al., 1999; Donner et al., 1993).
CMV infection in the first or second trimester of pregnancy typically results in an outcome for the fetus or the newborn that is less favorable than CMV infection in the third trimester (Stagno et al., 1986). Early detection, especially in the first trimester or early in the second trimester, would facilitate the option to terminate the pregnancy. Therefore, early detection of fetal CMV infection is important.
CMV infection is typically detected by assaying for CMV in amniotic fluid. However, in order to reliably detect CMV in amniotic fluid, the amniocentesis must be performed after 21 weeks of gestation (i.e., almost the end of the second trimester), and at least six weeks after seroconversion (Grangeot-Keros et al., 2001).
Another problem with detecting CMV by amniocentesis is that detection of CMV in amniotic fluid merely differentiates infected from uninfected fetuses. This method for detecting CMV does not reveal the extent of infection in the fetus.
Accordingly, detecting CMV in amniotic fluid does not accurately determine fetal outcome. An accurate determination of fetal outcome is important in order to provide appropriate counseling for parents.
Commercial ELISA kits that measure CMV-specific antibodies have also been used to detect congenital CMV infection (Azam et al., 2001). The antigen used for detection of CMV-specific antibodies in the ELISA is typically either a specific structural protein (e.g., pUL32, pUL83, pUL80a) or a specific nonstructural protein (e.g., pUL57, pUL44).
These commercial ELISA kits are used in assays in which blood is taken from a patient at a time that corresponds to a point in the life cycle of the virus. Various viral proteins are produced in different amounts during the life cycle of the virus. The variation in amounts of viral proteins produced by the virus results in differences in the amounts of antibodies, produced by the patient, to the viral proteins.
Since the commercial ELISA kits measure the amount of antibodies to a specific viral protein, the result of the assay depends on when blood is obtained and on which protein the commercial kit uses as an antigen. Therefore, the correlation of results obtained with different commercial kits that measure CMV-specific antibodies is poor (Lazzarotto et al., 1992). Contradictory results may be obtained if a serum sample is tested with two different kits. The sensitivity of these commercial ELISA is reported to be only 20-70% (Hogge et al., 1993; Hohlfeld et al., 1991).
Therefore, there is an immediate need for a reliable method for detecting congenital CMV infection and for determining the extent of the infection in a fetus. In addition, since about 10% of asymptomatic newborns will suffer late sequelae of a CMV infection, there is a need to assess CMV infection in a neonate at the time of birth.
The above needs have been satisfied by providing a method for detecting the presence of CMV in a fetal sample. The method comprises obtaining a fetal sample from a human fetus of an expectant mother infected with CMV, and determining the presence of anti-hsp70 antibodies in the fetal sample. The presence of anti-hsp70 antibodies indicates the presence of CMV in the fetal sample.
In another embodiment, the invention provides a method for monitoring CMV infection in a human fetus. The method comprises obtaining a first fetal sample from the fetus of an expectant mother infected with CMV, determining the amount of anti-hsp70 antibodies in the first fetal sample, obtaining a subsequent fetal sample, and determining the amount of anti-hsp70 antibodies in the subsequent fetal sample. A change in the first sample compared to the subsequent sample indicates a change in the infection. Such changes permit monitoring of CMV infection in the fetus.
In yet another embodiment, the invention provides a method for detecting the presence of CMV in a cord blood sample of a neonate. The method comprises obtaining the cord blood sample and determining the presence of anti-hsp70 antibodies in the sample. The presence of anti-hsp70 antibodies in the sample indicates the presence of CMV in the cord blood sample.